Influenza is a highly infectious disease caused by rapidly mutating influenza viruses. It is easily transmitted and spreads around the world in seasonal epidemics, infecting 5-20% of the total population annually. According to the World Health Organization (WHO), 250,000-500,000 people die annually of influenza-related causes during seasonal outbreaks. In the USA alone, more than 200,000 people are hospitalized with seasonal influenza in a typical year. Influenza infection may be mild, moderate or severe, ranging from asymptomatic through mild upper respiratory infection and tracheobronchitis to a severe, occasionally lethal, viral pneumonia. The infection is associated with pulmonary and cardiovascular complications leading to high morbidity and mortality rates, affecting mainly at-risk populations such as toddlers, elderly and individuals with chronic medical conditions.
Of the three types of influenza viruses, Influenza A and Influenza B are responsible for approximately 80% and 20% of influenza disease in humans, respectively, while influenza C viruses do not infect humans. Influenza A viruses are characterized by many sub-strains and by species specificity and are considered to be the major cause of widespread seasonal epidemics and of pandemics, due to the frequent antigenic drifts and shifts of the Hemagglutinin (HA) and Neuraminidase (NA) surface proteins. Following antigenic changes, infection via virus strains which are unrecognized by the immune system may result in a reduced immune response by the infected individual, where more significant changes will yield less effective stimulation of the body's immune defenses. Antigenic drifts or shifts can trigger respective influenza epidemics or pandemics, as experienced with the recent Avian and Swine Flu pandemic strains.
To date, commercially available influenza vaccines contain influenza A and B antigens that are annually selected according to predictions of the strains to be most prevalent during the peak influenza season. However, due to the mismatch between the strains included in the vaccine and those actually circulating, these strain-specific vaccines often have relatively poor clinical efficacy. In addition, such immunization methods require preparation of new vaccine formulations on an annual basis. Thus, a vaccine recognizing multiple virus strains would be more cost effective and would further increase patient compliance and enhance global health prospects.
Commercial influenza vaccine compositions currently in use are aqueous solutions comprising typically phosphate, sodium, potassium and/or calcium buffers with additions of Triton, Tween, α-tocopheryl hydrogen succinate and/or other additives or excipients. FLUMIST™ (MedImmune), a live attenuated vaccine for influenza that presents the surface antigens of seasonal flu, is supplied as a solution comprising about 0.05 M arginine, 0.188 mg monosodium glutamate, 2 mg hydrolyzed porcine gelatin, 13.68 mg sucrose, 2.26 mg dibasic potassium phosphate, and 0.96 mg monobasic potassium phosphate.
PCT International Publication WO 2009/016639 to some of the inventors of the present invention discloses influenza multi-epitope polypeptides and vaccines comprising a plurality of influenza virus peptide epitopes wherein each epitope is present at least twice in a single polypeptide.
The Multimeric-001 (M-001) vaccine consists of nine conserved linear epitopes arranged as three repetitions of each and prepared as a single, recombinant polypeptide expressed in Escherichia coli (E. coli). These epitopes are common to the vast majority of influenza virus strains, regardless of their antigenic drifts and shifts. Consequently, M-001 is expected to provide immunity-based protection against future virus strains as well. Multimeric-001 vaccination leads to efficient cross-strain recognition and protection despite variations in the outer proteins of each strain.
The significant results obtained with various animal models and the safety parameters observed in the repeated toxicology study have paved the way toward, and provided the foundation for, clinical trials in humans. Phase I/II and Phase II clinical trials assessing the safety and immunogenicity of M-001 in adult and elderly volunteers were completed (Atsmon et al., 2014, Vaccine 32, 5816-5823). Doses of 125-500 μg adjuvanted or non-adjuvanted vaccine, in PBS, proved safe and well tolerated. Potential Multimeric-001 vaccine-related toxicity was evaluated in GLP toxicology studies. Both M-001 vaccine formulations (adjuvanted and non-adjuvanted) repeatedly IM administered at the maximal human dose, proved to be safe.
WO 2012/114323 to some of the inventors of the present invention provides a method of improving the protective effect of a seasonal or pandemic influenza vaccine by administering to a subject, prior to or together with the influenza vaccine, a multimeric influenza polypeptide comprising multiple copies of plurality of influenza virus peptide epitopes.
Formation of inclusion bodies (IBs) frequently occurs when heterologous proteins are expressed in E. coli, and recovery of the active recombinant protein often requires refolding into its active structure. Wingfield, P. T. (Current Protocols in Protein Science. 2003, 30:6.1.1-6.1.37) reviewed the purification of recombinant proteins produced in E. coli. 
Arginine is used for refolding and purification of proteins obtained from IBs and appears to be effective for a variety of proteins differing in chemical and physical properties. The role of arginine in protein refolding, solubilization and purification was reviewed by Tsumoto et al., (Biotechnol Prog. 2004 September-October; 20(5): 1301-8; Schneider et al., (J Phys Chem B. 2011 Jun. 9; 115(22):7447-58)).
An anti-aggregation effect and stabilizing effect of arginine was previously described. For example, Lyutova et al., (Biotechnol Prog. 2007 November-December; 23(6):1411-6) studied the effect of low concentrations of arginine (1-10 mM) on protein aggregation, when protein aggregation is induced by transition from a folded state by heating or by addition of dithiothreitol (DTT).
Low-molecular-weight additives, such as L-arginine, have been suggested to enhance renaturation yields by inhibiting intermolecular hydrophobic interactions that lead to precipitation. Ho et al., (Protein Sci. 2003, 12, 708-716) demonstrated that L-arginine suppresses aggregation by increasing protein solubility.
Production of a protein in the form of a pharmaceutical suspension is generally favored as a result of one or more of the following reasons: the solubility of the polypeptide; the stability of the polypeptide; controlling or altering the release profile of the polypeptide.
Suspensions of protein micro- or nano-particles may be produced by a number of methods. Such particles may be initially produced as larger particles, followed by size reduction procedure by physical or chemical means. Some other approaches include crystallization, lyophilization, spray-drying and supercritical fluid particle formation or desolvation.
WO 2009/015736 discloses a process of production of a purified recombinant GDF-5 related protein comprising treatment of the inclusion bodies with a denaturing solubilization buffer comprising L-arginine.
WO 2012/054679 discloses methods for purifying a recombinant protein from a mixture comprising the recombinant protein and inclusion bodies, the method comprising: a) solubilizing the mixture comprising the recombinant protein with associated inclusion bodies with a solubilization buffer comprising ethanolamine, arginine, EDTA, urea and DTE.
US 2003/0199441 relates to a method to produce renatured procollagen propeptides wherein inclusion bodies produced in E. coli are dissolved in a 0.5 to 8 M denaturing buffer which is then added dropwise into a limited dilution buffer that is buffered around neutral pH and contains L-arginine in a final concentration between 200 to 1,000 nM and a disulfide bridges-reducing coupled redox system, and then the buffer mixture is dialyzed against a physiological buffer that contains L-arginine at a final concentration of 50 to 200 nM and a disulfide bridges-reducing coupled redox system and later against a physiological buffer that contains a disulfide bridges-reducing coupled redox system and finally against a physiological buffer.
US 2004/0137588 describes a method of purifying polypeptides from a biological sample subjecting the polypeptide to refolding conditions in the presence of arginine.
It would be advantageous to have stable suspensions of microparticulate polypeptides having a uniform size distribution for use in improved multimeric-multiepitope influenza vaccines. Efficient production processes of such compositions are also required.